Studies from the laboratory of the PI have focused on elucidating the components, mechanisms, and roles of bioactive sphingolipids, especially ceramide with its emerging roles in regulating cell growth, differentiation, senescence, and inflammation. However, the study of these pathways has been complicated by their multiplicity, metabolic interactions, sub cellular compartmentalization, and lack of molecular tools. These considerations led us to the overall hypothesis that the specific cellular functions of ceramide are dictated by the sub cellular site of ceramide generation which in turn depends on which of the enzymes of ceramide metabolism regulates its site-specific formation. In this proposal we will focus on the neutral sphingomyelinase (SMase) pathway and address the following aims: 1) To define cellular mechanisms of regulation of nSMase2. We will investigate the specific hypothesis that defined signaling pathways lead to the activation/regulation of n-SMase2 in response to TNF action. This will be approached by defining the regulation of translocation of nSMase2 by TNF, and determining the cellular mechanisms that regulate this translocation. 2) To determine molecular and biochemical mechanisms of activation of nSMaseZ. We will determine the mechanisms by which nSMase2 interacts with phosphatidylserine, an activating lipid, and we will determine the mechanisms of phosphorylation and palmitoylation and their roles in activation and translocation of nSMase2. 3) To determine the role of nSMase2 in generating a specific 'pool'of ceramide with specific downstream functions. We will test the specific hypothesis that nSMase2 acts on a distinct pool of sphingomyelin that resides in the inner leaflet of the plasma membrane, leading to the corresponding subset of ceramide, with its own specific functions. Taken together, these results should provide a foundation for the study of molecularly-distinct sphingolipid-mediated pathways of cell regulation. Moreover, molecular studies on nSMase2 would also impact our understanding of the mechanisms of an emerging and novel family of phospholipid phosphodiesterases. These pathways are of direct significance to the biology of stress responses and specifically to cancer biology and inflammation.